Protein, Enzyme, and Biological Water Dynamics: 2D Vibrational Echo Spectroscopy

描述（由申请人提供）：研究计划研究一系列相关的问题，涉及蛋白质、酶、酶-底物结合的动力学和动态结构关系，以及生物水的性质。该提案分为两个相关的部分。第一部分讨论蛋白质和酶动力学。第二部分涉及生物水的性质及其对生物系统的影响。主要的实验工具是超快二维红外振动回波光谱和其他超快红外方法。振动回声实验类似于二维核磁共振，除了它们直接在其他方法无法达到的时间尺度上检查生物系统的结构/机械自由度。结合分子动力学模拟和其他理论方法分析了2D-IR结果。在我们最初成功地利用2D-IR光谱阐明底物结合与蛋白质动力学之间关系的基础上，新的方法将应用于如何通过外源配体在活性位点的结合来修饰蛋白质结构动力学的重要问题。CO和叠氮化物探针将被选择性地引入几种过氧化物酶的活性位点。关键结构基序和蛋白质功能之间的相互作用将在几个系统中进行检查。神经红蛋白（Ngb）是一种具有单二硫键的血红素蛋白，参与调节蛋白质的氧结合亲和力。Ngb内发生的结构和动态转变将通过生物化学和诱变破坏二硫键来探测。蛋白质功能和结构转化之间的关系也将在其他系统如硝基磷蛋白中进行研究。将采用最近开发的方法，在活性位点和蛋白质的特定位置选择性地引入蛋白质动力学的位点特异性探针。通过研究细胞色素c （cyt c）在水和溶胶-凝胶纳米孔环境中的变性，探讨纳米限制对蛋白质展开的影响。以胍、盐酸、尿素、甲醇和pH为化学变性剂的变性研究将探讨熔融球态的动力学性质。由于纳米尺度的限制和与生物大分子的密切接触，生物水的行为与普通水明显不同。我们对纳米级水动力学的成功2D-IR测量将扩展到磷脂和非离子表面活性剂的反胶束。水在蛋白质界面上的动力学将通过将蛋白质限制在反胶束中并观察水氢键动力学来确定。由于水与跨膜蛋白和其他生物分子的相互作用，膜表面的水性质在生物过程中起着重要的作用。水在模型磷脂膜表面的动力学和相互作用将使用2D-IR光谱进行研究。水在gramicidin（一种跨膜质子通道蛋白模型）多层结构中的动力学将通过超快红外光谱测定。复杂生物分子（如蛋白质和酶）的结构动力学决定了它们如何发挥其生物学功能。该项目使用先进的红外激光技术直接检测生物分子结构动力学，以及生物分子与周围介质（特别是生物环境中的水）的相互作用如何影响结构动力学。该方法建立在以前的成功应用和最先进的超快红外激光实验的发展。

DESCRIPTION (provided by applicant): Research is proposed to study an interrelated set of problems involving the dynamics and dynamics-structure relationships of proteins, enzymes, enzyme-substrate binding, and the nature of biological water. The proposal is organized into two related parts. The first part discusses protein and enzyme dynamics. The second part involves the properties of biological water and its impact on biological systems. The principal experimental tools are ultrafast 2D-IR vibrational echo spectroscopy and other ultrafast IR methods. The vibrational echo experiments are akin to 2D-NMR except that they directly examine the structural/mechanical degrees of freedom of biological systems on time scales not accessible by other methods. The 2D-IR results are analyzed in conjunction with molecular dynamics simulations and other theoretical approaches. Building on our initial successful work in elucidating the relationship between substrate binding and protein dynamics with 2D-IR spectroscopy, novel approaches will be applied to the important question of how protein structural dynamics are modified by binding of exogenous ligands in the active site. CO and azide probes will be introduced selectively within the active site of several peroxidases. The interplay between key structural motifs and protein function will be examined for several systems. Neuroglobin (Ngb) is a heme protein with a single disulfide bond that is implicated in modulating the protein oxygen binding affinity. Structural and dynamic transformations that occur within Ngb will be probed by biochemically and mutagenically disrupting the disulfide bond. The relationship between protein function and structural transformation will also be examined in other systems such as nitrophorins. Recently developed methodology to introduce site-specific probes of protein dynamics selectively within the active site and at specific locations in the protein will be employed. The effects of nanoscopic confinement on protein unfolding will be probed by studying the denaturation of cytochrome c (cyt c) in aqueous and sol-gel nanopore environments. Denaturation studies with guanidine HCl, urea, methanol, and pH as chemical denaturants will probe the dynamical properties of molten globule states. Biological water differs markedly from bulk water behavior because of the effects of nanoscopic confinement and intimate contact to biological macromolecules. Our successful 2D-IR measurements of the dynamics of nanoscopic water will be extended to reverse micelles with phospholipid and non-ionic surfactants. The dynamics of water at protein interfaces will be determined by confining proteins in the reverse micelles and observing the water hydrogen bond dynamics. Water properties at membrane surfaces play an important role in biological processes because of water's interaction with transmembrane proteins and other biomolecules. The dynamics and interactions of water at the surfaces of model phospholipids membranes will be studied using 2D-IR spectroscopy. The dynamics of water in gramicidin, a model for transmembrane proton channel proteins, in multibilayers will be determined via ultrafast IR spectroscopies. PUBLIC HEALTH RELEVANCE The structural dynamics of complex biological molecules, such as proteins and enzymes, determine how they perform their biological functions. This project is using advanced infrared laser techniques to directly examine biomolecular structural dynamics and how biomolecule interactions with the surrounding medium, particularly water in biological environments, influence structural dynamics. The methodology builds on previous successful applications and developments of state-of-the-art ultrafast infrared laser experiments.